This application claims foreign priority benefit of European Patent Application No. 99811084.5 filed Nov. 25, 1999.
The present invention in the field of commercial fish farming for meat production relates to the control of parasites, which attach themselves to the skin of fish. To be more precise, it relates to the control thereof using the active substances named in claim 1, preferably by injection.
Fish farming, particularly when used to produce meat, operates nowadays on a large scale in so-called fish farms, where numerous fish are farmed in a confined area until they are ready for slaughter or for sale. As with any intensive livestock farming, in this case also, diseases and parasite infestation can lead to substantial losses and thus to drastic financial losses. As well as diseases caused by microorganisms such as Protozoa or by fungi, ecto-parasites that are customarily called sea lice, play a particularly decisive role.
Sea lice have absolutely nothing to do with insects, but as described in more detail below, belong to the fish-parasitic crustacea. There are in particular two members of the class of Copepodae [hoppers], which lead to substantial losses in yield, namely Lepeophtheirus [Lepeophtheirus salmonis] and Caligus [Caligus elongatus]. Primarily, they are popularly known as sea lice. They are easily recognised by their brown horseshoe-shaped shell, with Lepeophtheirus being considerably larger than Caligus.
These sea lice bite the fish firmly and damage it by eating the scales, the cell tissue and the mucous membrane. In the case of severe infestation, these parasites even penetrate into deeper layers of tissue. The immune system of the fish is weakened, leading to secondary infections and an excessive accumulation of water in the tissue. Frequently, the excessive parasite infestation leads to increasing tissue damage and, due to natural or artificial ultraviolet radiation or due to osmotic shock or the secondary infection, finally leads to death of the fish. Even with a light infestation, the fish lose body weight and only reach the right size for slaughter very slowly, if at all. In addition, infested fish have an unpleasant appearance and are not accepted by bulk buyers and end consumers.
By now, the sea louse can be found on almost all fish farms. Mortality rates based on infestation by sea lice of more than 50% have been reported by Norwegian fish farms. The extent of damage depends on the season and environmental influences, such as the salt content of the water and the average water temperature. In an initial phase, the sea louse infestation is observed by the parasites attached to the fish, and later onxe2x80x94more significantlyxe2x80x94by the damage to the skin and the tissue. The greatest damage is observed on smolts which are in that period of life in which they migrate from fresh water to sea water. The whole situation is made worse by the specific conditions in the fish-breeding farms, where often salmon of different years, but the same class of weight, are kept together; soiled nets or cages are used; high salt concentrations are found; little running water flows through the nets and cages, and the fish are kept in a very small area.
Fish farmers who are confronted with these parasite problems have to accept substantial financial losses and additional costs. On the one hand, their fish are weakened and damaged by the lice, which leads to lower rates of weight increase; and on the other hand secondary infections have to be kept in check with expensive medicines and labour-intensive measures. In many cases, the goods can no longer be sold, as the damaged fish deter the consumers. For salmon breeders, this problem of lice infestation may threaten their existence.
The greatest damage is produced by Lepeophtheirus, since even a few parasites cause vast tissue damage. The life cycle of Lepeophtheirus consists basically of two larval stages living freely in water [Naupilus and Copepodia stages], four Chalimus stages, one pre-adult and the actual adult stage. The Chalimus and adult stages are host-dependent.
The most dangerous, since they produce the greatest damage, are all the fish-parasitic stages of sea louse, in particular the actual adult stages.
In the meantime, a series of chemical substances have been used against these sea lice with more or less success, e.g. trichlorfon [dimethyl-2,2,2-trichloro-1-hydroxyethyl-phosphonate], which requires concentrations of 300 ppm in salt water, and dichlorvos [2,2-dichloroethenyl-dimethylphosphate], which is effective from 1 ppm. A disadvantage of these preparations is the relatively high application rates, and the environmental contamination associated therewith, which also applies all the more because of the relatively high half-life periods. Other more selective substances that are used successfully are described e.g. in EP-497,343, EP-590,425, EP-781,095 and WO 97/21350. The fish are usually treated orally, e.g. through the food, or topically, i.e. externally by means of bath treatment, for example in a xe2x80x9cmedicinal bathxe2x80x9d into which the fish are placed and kept for a period [minutes to several hours], e.g. transferring from one breeding tank to another. If there is no possibility of transferring the fish into a special tank, normally temporary or long-lasting treatment of the habitat of the fish takes place, e.g. in net cages, whole ponds, aquariums, tanks or basins, in which the fish are kept. In individual cases, treatment also takes place parenterally, e.g. by injection, especially if this is a hand-picked specimen for further breeding or ornamental fish as part of a hobby.
Although there are substances that show good efficacy against fish parasites, there is a need for further active substances that are more effective, can be tolerated by the underwater flora and fauna, or can be handled without problems by the breeder. Of course, the shelf life and stability in feed mixtures are also important. In addition, practicable application methods are desired, which save time and energy or further reduce environmental contamination. In this rapidly expanding industry, the above factors play an ever increasing role and can be crucial to commercial success.
WO 92/06599 describes the administration of oral compositions to fish and depicts this as an especially advantageous method compared with the labour-intensive and complex injection of individual fish. An injection is described as a particular stress factor, which at the very least leads to a temporary reduction in growth.
In contrast to this, it has now surprisingly been established that, with appropriate handling, the injection can have significant advantages over the other types of administration, if it is used in mass breeding using suitable apparative measures. It was established that the injection nowadays does not have to be restricted to specific cases, e.g. for especially expensive breeding and ornamental fish or for individually selected sick fish, but can be carried out with relatively little manual effort and using little time, even for whole schools of fish, without exposing the fish to exceptional stress. As will be shown in the following, whole schools of hundreds or thousands of fish may be treated in an almost stress-free manner, giving rise to quite significant advantages. The present invention accordingly relates also to the treatment of whole schools of fish, i.e. to commercial fish breeding for meat production, which is also known by the name xe2x80x9cfish-farmingxe2x80x9d. Under no circumstances should this be confused with the known occasional treatment of individual sick fish or with individual experiments to establish the efficacy of a potential active ingredient.
The conventional treatment processes that are successful per se of course also have their down side. The serious disadvantages of the current water treatment method are that the active substance and the remaining formulation excipients are not specifically targeted to the fish or the parasites, but extend inevitably over the whole habitat of the fish and may interact with the underwater flora and fauna or in detrimental cases may even reach the drinking water. Therefore, either extremely selective active substances must be used, or the fish must be transferred to closed basins or tanks for the duration of treatment and treated there whilst screened from the environment. However, the problem that remains after successful treatment is the removal of the water from the basin or tank. In addition, because it is inevitable that the active ingredient is diluted, a drastic excess dosage must be used for it to reach the parasites in a sufficiently active concentration. Fish food, in which the active ingredient has been incorporated, is fraught with the same problems.
Another great disadvantage of conventional treatment methods is that the active substance remains in the water over rather long periods of time in a greatly diluted and thus sub-lethal dose, which can substantially encourage resistance to build up in the target parasites.
On the other hand, if the active substance or a corresponding preparation is injected, the dose can be precisely coordinated to the body weight and substantially counteract the formation of resistance. In the case of fish being bred for meat production, this is especially easy to accomplish, as the whole population is of the same age and weight. Using the injection method, there is no overdosing or underdosing, and the environment remains as unharmed as possible, since it has no contact whatsoever with the active ingredient.
Of course, each fish could be treated by hand using an injection syringe. In fact, this would be time-and labour-intensive and certainly a venture associated with a certain amount of stress for the fish.
It has now however surprisingly been found that the above problem can be solved much more elegantly in commercial fish farming by treating a whole school of fish with an automatic or semi-automatic injection device. What is essential to this method is that the fish to be treated are guided in single file past an automatic inoculation device which administers the correct single dose to each fish, based on body weight and severity of infestation. In a preferred embodiment, this takes place by forcing the school of fish through a narrow passage, e.g. a narrow channel or trough, in which there is an addition constriction, for example a small elevation or another type of additional obstacle, so that the fish have to cross this narrow passage in single file and have to briefly stop at the said additional obstacle. If this channel is preferably kept so shallow that the dorsal area of the fish remains just below the surface of the water or even protrudes from the water a little, the fish is forced to navigate the narrow passage slowly. If the flow of fish is additionally checked or stopped by a further obstacle, each individual fish can be administered with the optimum dose within a short time, e.g. either by hand using an injection syringe or preferably with an appropriate injection device, e.g. an inoculating gun, thus making the procedure semi-automatic. If necessary, one or more grids or other obstacles may be provided across the flow, to slow down the progress of the school in the narrow channel, so that no fish is overlooked or can pass through untreated. Using a mechanical, optical, thermal or movement sensor, treatment may be further automated, so that each fish that passes the sensor makes a contact, which brings the injection device into an appropriate position and carries out the injection. Through these measures, the actual proportion of manual work and the duration of the treatment procedure are reduced to a minimum and the stress for the animals to be treated is kept to an acceptable limit.
It has been demonstrated that the fish rapidly overcome the short shock phase and no longer show any stress reactions even one day after treatment. At latest two days later the fish show absolutely normal eating and group behaviour and their weight shows the usual increase. In addition, the targeted dosage which is spread evenly over the population ensures that the parasite infestation is reduced in a totally balanced manner over the entire population and sets in more quickly than in the case of water treatment. When treating the water, a balanced reduction is only attainable by means of massive overdosing.
This injection method can be used not only as a curative method, but also, advantageously, prophylactically. The latter is even preferable, as the vitality of the fish is maintained and there is no damage from the parasites that has to be cured. In addition, this type of prophylactic treatment is cheaper because of the low dosage, especially when compared with the water treatment method, and moreover is very environmentally friendly.
In the context of the present invention, injection is understood to mean not only all measures which are carried out using a needle, but also needleless methods, in which the active substance is fired through the skin using pressure, e.g. from an inoculation gun as used in human or animal medicine. The injection according to the invention provides administration through the skin, primarily into muscle or fat tissue.
The method according to the invention of controlling sea lice in commercial fish farming consists in applying an amount of an appropriate active substance that is effective against sea lice not through the medium water, but percutaneously and therefore directly to each member of a school of fish. Percutaneous is understood to mean preferably the above-mentioned types of injection.
The preferred embodiments of the present invention include, interalia:
A method of controlling sea lice in commercial fish farming, characterised in that an amount of an appropriate active substance that is effective against sea lice is administered individually to each fish either manually, semi-automatically or by an automated injection device, whereby semi-automatic and especially automatic administration are preferred.
A further important aspect of the present invention consists in the usage of an automated injection device for administering a dosage of a substance that is effective against sea lice, the dosage being effective per single fish, in a method of controlling sea lice on fish in commercial fish farming.
In accordance with the invention, the described method is used to advantage for the control of sea lice, which from a botanical aspect belong to the fish-parasitic crustacea. These include inter alia the Copepodae [hoppers] of the genera Ergasilus; Bromolochus; Chondracaushus; Caligus [Caligus curtus, Caligus elongatus]; Lepeophtheirus [Lepeophtheirus salmonis]; Elythrophora; Dichelestinum; Lamproglenz; Hatschekia; Legosphilus; Symphodus; Ceudrolasus; Pseudocycmus; Lemaea; Lemaeocera; Pennella; Achthares; Basanistes; Salmincola; Brachiella; Epibrachiella; Pseudotracheliastes; and the familes: Ergasilidae; Bromolochidae; Chondracanthidae; Calijidae; Dichelestiidae; Philichthyidae; Pseudocycnidae; Lemaeidae; Lemaepotidae; Sphyriidae; Cecropidae, as well as the Branchiuriae [crabs] of the family Argulidae and the genera Argulus spp.; and the Cirripediae [barnacles] and Ceratothoa gandichaugii. 
The targets of the treatment according to the invention are commercial fish of all ages, which live in freshwater, sea water and brackish water, e.g. carp, eel, trout, whitefish, salmon, bream, roach, rudd, chub, sole, plaice, halibut, Japanese yellowtail [Seriola quinqueradiata], freshwater eel [Anguilla japonica], red seabream [Pagurus major], sea bass [Dicentrarchus labrax], grey mullet [Mugilus cephalus], pompano, gilthread seabream [Sparus auratus], Tilapia spp., Cichlidae species such as Plagioscion, Channel catfish.
The treatment according to the invention is especially suitable for breeding salmon. The term salmon in the context of the present invention includes all members of the family of Salmonidae, especially those of the subfamily Salmonini and preferably the following species: Salmon salar[Atlantic salmon]; Salmon trutta[brown or sea trout]; Salmon gairdneri[rainbow trout]; as well as the Pacific salmon [Oncorhynchus]: Oncorhynchus gorbuscha; Oncorhynchus keta; Oncorhynchus nekra; Oncorhynchus kisutch, Oncorhynchus tshawytscha und Oncorhynchus mason; also included, however, are the species modified by breeding, e.g. Salvelinus species and Salmo clarkii. 
Particularly preferred targets of the present invention are the Atlantic and Pacific salmon and the seawater trout.
In modern salmon and trout farming, young fish at the smolt stage are transferred from freshwater basins to seawater cages [salt water]. These are usually cubic, rectangular or even round cages consisting of a basic metal frame surrounded by a relatively fine-mesh net. These cages are lowered into the sea to ca. 9/10 and anchored, so that they are accessible from the top. The treatment process according to the invention can be employed particularly well using this transfer method. This prevents the active substance from being released into the sea and having an adverse affect on other sea creatures.
In another variant, the fish are kept in seawater basins or tanks of different forms. The cages are arranged in bays in the sea in such a way that the current constantly passes through and a sufficient oxygen supply is assured. The salt water in the seawater tanks is also kept in circulation with a supply of oxygen. In the artificial environment, the fish are fed until they are sufficiently matured and can be used commercially as food or can be sorted for further breeding. Here also, with single or multiple relocation, the injection process according to the invention can be used successfully.
In these fish breeding farms, there is extremely intensive cage maintenance. The density of fish reaches the order of 10 to 25 kg fish/m3. With this monoculture and the extremely high fish concentrations, together with the usual stress factors, the fish caught are generally found to be considerably more susceptible to diseases, epidemics and parasites than the free-living members of the same species. For treatment against sea lice by the process according to the invention, the relocation method to other cages may be used, whereby the fish are shepherded through the initially-described narrow passage to the injection device.
The total dose of injection for the same active ingredient may vary from one species of fish to another and even within one species, since it depends inter alia on the weight, the age and the constitution of the fish. Furthermore, the dose depends on the activity of the active ingredient employed.
Advantageous doses are between 10 and 100 mg/kg body weight, preferably between 20 and 70 mg/kg body weight.
As injection preparations according to the invention, the active ingredients are normally not applied in pure form, but preferably in the form of a composition or preparation which contains, in addition to the active ingredient, application-enhancing constituents or formulation excipients, whereby such constituents are beneficial to the fish. In general, beneficial constituents are the formulation excipients for injection preparations which are physiologically tolerated by humans and animals and are known from pharmaceutical chemistry.
Such injection compositions or preparations to be used according to the invention usually contain 0.1 to 99% by weight, especially 0.1 to 95% by weight, of a substance that is active against sea lice, e.g. a compound of formula (I), and 99.9 to 1% by weight, especially 99.9 to 5% by weight, of a liquid, physiologically acceptable excipient, including 0 to 25% by weight, especially 0.1 to 25% by weight, or a non-toxic surfactant and water.
Whereas it is preferred to formulate commercial products as concentrated injection formulations, the end user will also use dilute formulations.
The formulations suitable for injection are for example aqueous solutions of the active ingredients in water-soluble form, e.g. a water-soluble salt, in the broader sense also suspensions of the active ingredients, such as appropriate oily injectable suspensions, whereby e.g. to delay the release of active ingredient (slow release), suitable lipophilic solvents or vehicles are used, such as oils, e.g. sesame oil, or synthetic fatty acid esters, e.g. ethyl oleate, or triglycerides, or aqueous injectable suspensions containing viscosity-increasing agents, e.g. sodium carboxymethyl cellulose, sorbitol and/or dextran, and where appropriate stabilisers. Oil-containing formulations with delayed release of active ingredient are called depot preparations here and hereinafter, and they belong to the preferred embodiments of the present invention, since, especially in the case of prophylactic administration, they are able to protect the fish for long periods from an infestation by the sea lice.
In the following examples, if not expressly stated to the contrary, the term xe2x80x9cactive ingredientxe2x80x9d represents 1-[4-chloro-3-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenyl]-3-(2,6-difluoro-benzoyl)urea.